High throughput digital quantification of mRNA abundance in primary human acute myeloid leukemia samples
Jacqueline E. Payton, … , Mark A. Watson, Timothy J. Ley
Jacqueline E. Payton, … , Mark A. Watson, Timothy J. Ley
Published May 18, 2009
Citation Information: J Clin Invest. 2009;119(6):1714-1726. https://doi.org/10.1172/JCI38248.
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Technical Advance Hematology

High throughput digital quantification of mRNA abundance in primary human acute myeloid leukemia samples

Abstract

Acute promyelocytic leukemia (APL) is characterized by the t(15;17) chromosomal translocation, which results in fusion of the retinoic acid receptor α (RARA) gene to another gene, most commonly promyelocytic leukemia (PML). The resulting fusion protein, PML-RARA, initiates APL, which is a subtype (M3) of acute myeloid leukemia (AML). In this report, we identify a gene expression signature that is specific to M3 samples; it was not found in other AML subtypes and did not simply represent the normal gene expression pattern of primary promyelocytes. To validate this signature for a large number of genes, we tested a recently developed high throughput digital technology (NanoString nCounter). Nearly all of the genes tested demonstrated highly significant concordance with our microarray data (P < 0.05). The validated gene signature reliably identified M3 samples in 2 other AML datasets, and the validated genes were substantially enriched in our mouse model of APL, but not in a cell line that inducibly expressed PML-RARA. These results demonstrate that nCounter is a highly reproducible, customizable system for mRNA quantification using limited amounts of clinical material, which provides a valuable tool for biomarker measurement in low-abundance patient samples.

Authors

Jacqueline E. Payton, Nicole R. Grieselhuber, Li-Wei Chang, Mark Murakami, Gary K. Geiss, Daniel C. Link, Rakesh Nagarajan, Mark A. Watson, Timothy J. Ley

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Figure 7

The NanoString-validated, 33-gene M3-specific signature reliably identifies M3 samples, including those with normal cytogenetics and/or ambiguous morphology.

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The NanoString-validated, 33-gene M3-specific signature reliably identif...
PCA plots of the validated gene expression data demonstrate a clear separation of M3 t(15;17)-positive samples (red) from other FAB subtypes (gray). (A) Data from the Washington University AML discovery set, including 15 M3 and 62 M0, M1, M2, and M4 AML samples (1). The PCA plot shows clustering of all M3 samples with the PML-RARA rearrangement, but not of 1 sample with an M3 morphological diagnosis, normal cytogenetics, and negative FISH that did not respond to ATRA therapy (blue). (B) NanoString nCounter expression data were sufficiently robust to separate 11/11 of M3 t(15;17)-positive samples from other FAB subtypes. (C) M3 samples from a published dataset (GSE6891) formed a distinct cluster separate from other FAB subtypes (total of 325). M3s with t(15;17) that were missed by routine cytogenetics (yellow) and a t(15;17)-positive sample morphologically classified as M2 (green) were also assigned appropriately to the M3 cluster. (D) A total of 19/20 M3s with t(15;17) from a CALGB sample set clustered separately from 73 other FAB subtypes.